The regulation of transcription is a key step in many biological processes in eukaryotic cells, including control of cell growth and division, differentiation of tissues and development of organs, and response to extracellular signals and environmental conditions. Transcriptional regulation is also critical to the temporally-coordinated expression of the genetic programs of many eukaryotic viruses, including SV40, adenoviruses, HIV, and herpesviruses. The long-term objective of this project is to understand the molecular mechanisms of transcriptional activation. Lytic infection by herpes simplex virus represents a useful model system for this objective, in that a component of the HSV virion (termed VP16) specifically activates transcription of the viral immediate- early genes. The activation domain of VP16 is among the most potent known, and thus has been widely adopted as a model for the study of transcriptional activation. This project has already made important contributions to models of the structure of transcriptional activation domains and their interactions with putative target proteins in the transcriptional machinery. A particular strength has been the use of techniques ranging from yeast genetics to fluorescence spectroscopy to test specific hypotheses. The specific aims for the next project period will continue to apply diverse methods to explore the mechanism of activation by VP16, and the role of this protein in the virion and the viral infectious cycle. Site-directed, systematic, and random mutational methods will be used to identify and characterize amino acids in the VP16 activation domain that are critical for its transcriptional function. The physical interactions of VP 16 with target proteins and the functional consequences of those interactions will be explored using biochemical assays. VP16 mutations will be introduced into the viral genome to evaluate the role of transcriptional activation by VP16 during lytic infection. The phosphorylation and nuclear localization of VP16 as a virion protein and its interaction with other virion proteins will be addressed. Finally, specific hypotheses will be tested regarding the identity and character of transcriptional activation domains of VP16 homologs from other alphaherpesviruses of medical and veterinary importance. The information gained from this project will be directly relevant to understanding transcriptional activation of RNA pol II by other regulatory proteins that mediate the wide range of biological processes noted above. This project will also provide knowledge necessary to explore antiviral strategies for human and veterinary use and for exploitation of HSV vectors as agents for selective gene therapy.